1. The Effects of Quinic Acid on Gene Expression of acetate mutants in Neurospora crassa S. Lawhorne, J.Griffith, J. Arnold, P. Brunson, R. Meagher and M. Case
Abstract: The qa gene cluster of N.crassa consists of 7 genes that dwell on linkage group VII.The genes play an important role in carbon metabolism (Tang et al). The purpose of this study was to test the hypothesis that when the organism is transferred from sucrose to Quinic Acid (QA) as its main carbon source, the organism would switch its metabolism from the carbon expensive Krebs Cycle to the Glyoxylate Cycle and continue to generate some of the energy from the Krebs Cycle. From this hypothesis we predict the quinate pathway feeds into the Glyoxylate Cycle, and the expression of genes affiliated with the Glyoxylate Cycle ( acu-3, acu-6, and acu-9) should increase in expression by being shifted to QA. Mutants in the Glyoxylate Cycle should grow poorly on QA. We conducted growth experiments of mutant and wild type strains, and an expression study of acu-3 mutant. The acu-3 mutant showed reduced growth on QA as predicted. The wild type allele of acu-3, showed an increase in expression thirty minutes after being shifted to QA, and there was a great increase in expression of the acu-3 mutant allele after six hours. We hypothesized that this increase in expression is due to the slow growth rate that the mutant endured. It is possible for N.crassa to survive on a poor carbon source, QA, by regulating the genes acu-3 and acu-9 and switching to the Glyoxylate Cycle.
The Glyoxylate Cycle is an alternative to the Krebs Cycle in N. crassa. This cycle allows the organism to take in carbon and produce energy. The enzymes isocitrate lyase (acu-3) and malate synthase (acu-9) provides a shortcut to the Krebs Cycle from isocitrate to malate. The genes acu-3 and acu-9 encode these enzymes. The part of the Krebs Cycle that is bypassed releases carbon dioxide in two areas. We hypothesize that an organism that does not have sufficient carbon, such as one using quinic acid, avoids this part of the Krebs Cycle. The figure is taken from the Neurospora Compendium by Perkins, Sachs, and Radford (2000).
Growth Tubes
In this experiment we measured the growth of the organism on sucrose(1.5%), quinic acid(0.3%), and acetate (4%). This was accomplished by marking the tubes that the organism was grown daily.The fungus grew from left to right all the way to the end of the tubes. The orange material in the tubes is conidia that the fungus produced as it grew. The progressing growth of the fungus was measured in millimeters per hour.
Sucrose (1.5%)
Quinic Acid (0.3%)
Acetate (4%)
Wild Type
(1.5%) Sucrose
Quinic Acid 0.3%
Acetate (4%)
acu-9KO
acu-6
acu-3KO
acu-3
Growth Tube Analysis of mutants and wild type
Viability Analysis of Mutants
In this experiment the acu-3 strain was grown in small tubes on sucrose (1.5%) for a couple of days. Then the sample was filtered for the conidia and diluted with water and placed in the incubator at 25 oC to grow. A seven day viability test on a Cellometer (Nexcelom Biosciences, Inc.) was conducted to see how long acu-3 could survive. The WT strain (data from P. Brunson) on the graph below was used as a control.
Shift Experiment from Sucrose (1.5%) to Quinic Acid (0.3%)
In this experiment an acu-3 strain was grown on sucrose (1.5%) for approximately 40 hours (due to slow growth) in 8 replicate flasks. These flasks were later harvested and shifted to QA for: 0 hr, 30 min, 60 min, 90 min, 2 hr, 4 hr, 6 hr, and 8 hr and subsequently harvested. When the organism is shifted from sucrose to quinic acid, the Glyoxylate Cycle begins regulating gene expression, and expression was measured on an acu-3 mutant by RT-PCR using ubiquitin as an endogenous control.
Neurospora crassa Crosses
E. Nottingham data on wild type (2011)
Current Acu-3 Data
This experiment included a cross between the WT(A) strain with acu-3KO and acu-9KO ,a. They were crossed with the WT(A) strain at the beginning of the summer, and they were placed in a cabinet in the dark at room temperature to grow. Then once spores were detected from the crosses they were plated, and colonies were isolated. The colonies were picked and inoculated on 4% acetate/Vogel’s Medium. The tubes that showed no growth on acetate were then tested on M hygromycin. After a couple of days three of the 46 isolates of the acu-3 cross had grown , and none of the acu-9 crosses grew on the hygromycin.
Summary: In the beginning of this study we saw that the acu-3 mutant grew at a much slower rate than the acu-6 and acu-9 mutants. We believe that the rate that the acu-3 mutant grew caused the great increase in expression at the six hour time point. When wild type was shifted from sucrose to quinic acid, an increase of expression was observed at the thirty minute time point since it grew at a faster rate than the acu-3 mutant. When qa genes are carbon starved as on QA, they will shift to the Glyoxylate Cycle. We concluded that acu genes are expressed at high levels when grown on QA as a sole carbon source.